Currently, quantification of small substances such as hormones, pharmaceuticals and peptides using an immunoassay is performed frequently. When a substance to be measured is a small substance, a quantification method referred to as a competitive inhibition method is used.
A measurement sensitivity in the competitive inhibition method depends on an affinity of an antibody to be used for an antigen, and generally when an antibody is raised against a small substance, it is known that it is difficult to obtain an antibody with high affinity. Also in the competitive inhibition method, specificity of an antibody is exerted based on reactivity of a type of antibody, and thus, it is necessary to acquire an antibody with high specificity, but it is difficult to acquire such an antibody. That is, a huge amount of effort is required for selecting an antibody that satisfies the sensitivity of the measurement and the specificity upon the measurement by the competitive inhibition method. Even if such an antibody can be selected fortunately, the competitive inhibition method is disadvantageous in that measurement accuracy and the measurement sensitivity are low and it is complicated to determine a competitive condition between a sample and a labeled antigen when a concentration of a substance to be measured is high or low.
The above disadvantage in the competitive inhibition method can be solved by a sandwich method of measuring using two antibodies that recognize different epitopes of an antigen. However, in the case of a small substance, there is only one epitope, and steric hindrance occurs between two antibodies. Thus, it is thought to be difficult to quantify the small substance using the common sandwich method.
A measurement method using an antibody that recognizes an antigen/antibody complex has been reported as a method for measuring a small substance using the sandwich method. In vivo methods using an animal (e.g., hybridoma methods) and in vitro methods (e.g., phage display methods) are known as methods of producing an antibody. For example, in Nonpatent Literature 1, it has been described that an antibody against a low molecular hapten (Δ9-tetrahydrocannabinol: MW 314.5)/antibody complex was successfully obtained by a mouse immunization method, and about 200 clones for the anti-mouse antibody (idiotype) antibody were established by repeating a fusion experiment 5 times, but only one clone was obtained as the antibody recognizing the complex.
Methods disclosed in Patent Literatures 1 and 2 and Nonpatent Literatures 1 to 4 are known as to methods of measuring a small substance by the sandwich methods.
Methods disclosed in Patent Literature 3 are known as to technologies of producing an antibody and antibody-producing cells.